The present invention relates to a monoclonal antibody which is not reactive with fibrinogen and is specifically reactive with a fibrin monomer. In an assay of fibrin in a body fluid, the monoclonal antibody is specifically reactive with a native fibrin monomer which is present in the body fluid without solubilizing, i.e. without cleavage of the fibrin, and is also specifically reactive with soluble fibrin. The invention also relates to an assay for detecting blood coagulation, without interference by various decomposition products of fibrin or of fibrinogen, using the monoclonal antibody.
A blood clot in a blood vessel is harmful to a living body, and thus detection of blood coagulation is useful for early diagnosis of various diseases.
In blood coagulation, fibrinogen is affected by active thrombin and consequently fibrinopeptide A, at the side of the N terminal of the xcex1-chain in the fibrinogen, is cleaved to form desAA-fibrin (desAA-Fbn), another name of which is fibrin I (Fbn-I).
Subsequently, fibrinopeptide B at the N-terminal of the xcex2-chain therein is cleaved to form desAABB-fibrin (desAABB-Fbn), another name of which is fibrin II (Fbn-II). The generic name xe2x80x9cfibrin monomersxe2x80x9d is given to desAA-fibrin and desAABB fibrin.
The formed fibrin monomer is then coagulated and crosslinked to produce fibrin clots. It is known that the period before clotting, when the fibrin monomer is present as an independent monomer in blood, is generally very short, and the fibrin monomer is associated with various proteins in blood, including fibrinogen, to be solubilized, that is, to form soluble fibrin.
C. E. Dempfle et al., (Blood coagulation and Fibrinolysis, 4: 79-86, 1993) reported an antibody obtained by causing thrombin to act on fibrinogen so as to cleave the fibrinopeptide A and then using the resultant N-terminal of the xcex1-chain as an immune source, and a method for measuring fibrin, using this antibody. However, the N-terminal of the xcex1-chain of fibrin is concealed by the interaction thereof with various blood proteins, including fibrinogen in blood, thus resulting in a drawback that the aforementioned antibody cannot react with soluble fibrin in blood.
H. Lill et al., (Blood coagulation and Fibrinolysis, 4: 97-102, 1993) suggested, as an assay for soluble fibrin in blood, a method of solubilizing soluble fibrin by chaotropic ions at a high concentration or the like to make the soluble fibrin into a fibrin monomer, and then measuring the resultant fibrin monomer. In this method, however, a reaction time is necessary for the solubilization for converting the soluble fibrin into the fibrin monomer. Thus, this measurement is not efficient. Moreover, a substance to be detected is diluted by the solubilization, resulting in a drawback that measurement sensitivity decreases.
G. Soe et al., (WO 95/12617; Blood 88(6): 2109-2117, 1996) suggested a method for assaying soluble fibrin without performing any pretreatment, such as solubilization for converting soluble fibrin into a fibrin monomer, and reported a monoclonal antibody which can directly recognize soluble fibrin for this method. The monoclonal antibody according to C. Soe et al., is a monoclonal antibody obtained by solubilizing fibrin clots by urea and then using the resultant urea-solubilized fibrin monomer as an immune source, and is a monoclonal antibody for recognizing a three dimensional structure change arising in the E-fraction of the fibrin monomer when the fibrin monomer generated in blood and fibrinogen form a complex. In the measuring method using this antibody, however, various proteins other than fibrinogen are present in blood, and therefore it is feared that the results of measurement are not accurate on account of the influence of proteins in blood (other than the fibrinogen) which associate with the fibrin monomer to form the complex. Moreover, in the measuring method using the present antibody, as the association-degree changes with the passage of time from the formation of the fibrin monomer to the generation of soluble fibrin, a change in its three-dimensional structure arises. For this reason, it is difficult to obtain a stable measurement because of change with the passage of time. Additionally, in the measurement for blood coagulation using the monoclonal antibody according to G. Soe et al., an epitope created by such a change in the three-dimensional structure does not emerge early in the blood coagulation, and further the antibody is not reactive with a native fibrin monomer generated in the blood.
Furthermore, in these conventional methods for assaying soluble fibrin, many of the antibodies used cross-react with fibrin decomposition products (XDP) in a body fluid, and thus it is difficult to say that they are specifically reactive with abnormal blood coagulation, in particular with an initial marker of diseases.
As described above, hitherto determination of blood coagulation in a living body, has used a monomer obtained by dissociating completed soluble fibrin by solubilization with a chemical agent or solubilized fibrin. There has not been known any assaying method using an antibody which can directly and simultaneously react with a fibrin monomer and soluble fibrin present in blood at the initiation of blood coagulation. An object of the present invention is to provide a monoclonal antibody for specifically detecting a native fibrin monomer produced at the initial stage of blood coagulation by the action of active thrombin, and detecting soluble fibrin simultaneously; a hybridoma which can produce the monoclonal antibody; and an immunoassay for assaying the initial stage of blood coagulation promptly, and with high sensitivity using the monoclonal antibody.
The present inventors have investigated use of a fibrin monomer analog in blood, as an immune source, to produce a monoclonal antibody which is not reactive with fibrinogen and can specifically and simultaneously recognize a native fibrin monomer and soluble fibrin. The native fibrin monomer is a fibrin monomer which is in a body fluid, in particular in blood, and is not solubilized. Herein, both the desAA-fibrin and the desAABB-fibrin are referred to as the fibrin monomer.
That is, the monoclonal antibody of the present invention is a monoclonal antibody which is specifically reactive with a native fibrin monomer, and at the same time is specifically and simultaneously reactive with a native, soluble fibrin wherein fibrinogen and a fibrin monomer are associated, even if a body fluid is used as a specimen, without being affected by the interaction of the fibrin monomer and admixed proteins other than the fibrinogen present in the body fluid.
The monoclonal antibody of the present invention is characterized by the aforementioned specific reactivity and not being reactive with various decomposition products of fibrin or fibrinogen, which are produced in a body fluid by cleavage by a plasmin.
The monoclonal antibody of the present invention makes it possible to directly assay a native-form substance to be detected, from the initial stage of blood coagulation, that is, the time when a fibrin monomer is produced, to the time when soluble fibrin is formed. This fact makes unnecessary solubilizing fibrin specimens for dissociation, as in the prior art. Therefore, operation efficiency, promptness and accuracy are improved in immunological assays. Furthermore, use of the monoclonal antibody of the present invention makes it possible to improve assay sensitivity since steps such as the step for solubilizing the fibrin specimen are unnecessary.
The method for producing a monoclonal antibody, according to the present invention, comprises fusing an antibody-forming cell obtained by using a fibrin monomer analog as an immune source to immunize an animal, and a myeloma cell by cell fusion, screening to obtain a hybridoma having antibody-forming ability exhibiting the desired reactivity, and establishing the hybridoma.
The reason why the fibrin monomer analog is used as an immune source is to obtain an antibody which is reactive with fibrin obtained by dissociating fibrinopeptide A in blood, that is, with both of a fibrin monomer and soluble fibrin in blood.
The reason why a native fibrin monomer itself is not used as an immune source in the production of the monoclonal antibody of the present invention is that the fibrin monomer in blood is associated with various blood proteins including fibrinogen, in blood, to form soluble fibrin, and consequently if the fibrin monomer itself is used an immune source, the possibility that an antibody specific to the fibrin monomer can be obtained is very low. Thus, the following method is considered: a fibrin monomer is purified from soluble fibrin and then the fibrin monomer is used as an immune source. For that, the soluble fibrin must be solubilized by a protein denaturant such as high-concentration urea or chaotropic ions. Since such protein denaturants change the three-dimensional structure of proteins, it is difficult to say that the resultant fibrin monomer is native. As a method for producing a fibrin monomer artificially without using any protein denaturant, a method using a coagulation inhibiting peptide is known. In this method, if thrombin is used to cleave fibrinopeptide A, which is a near native reaction, fibrinopeptide B is also cleaved. Namely, two types of complex formation inhibiting peptides become necessary for N-terminal cleavage sites in treatment of the fibrin monomer with thrombin, and thus the preparation becomes difficult.
The fibrin monomer analog in the present invention is defined as a substance which is similar to a fibrin monomer and is produced in body fluid by treating fibrinogen with an enzyme, a chemical agent or the like to dissociate fibrinopeptide. The number of amino acids of the dissociated fibrinopeptide is somewhat more or less than that of the fibrin monomer in body fluid, or the amino acids are substituted by other compounds. Preferably, the fibrin monomer analog in the present invention is fibrinogen treated with bathroxobin, which is a snake venom. Bathroxobin is specific in cleaving the peptide A of fibrinogen.
The reason why the monoclonal antibody of the present invention obtained by immunization by the fibrinogen treated with bathroxobin and cell fusion has the aforementioned specific reactivity would be presumably that even if by the reaction with an epitope present at a slightly internal site of the amino acid terminal of the active thrombin treated fibrinogen (that is, the fibrin monomer) the fibrin monomer is associated with the fibrinogen or other admixed proteins, the epitope is not masked.
In the method for producing the monoclonal antibody, according to the present invention, if the fibrinogen treated with the snake venom is used as is, it is associated with admixed proteins in a complex. If a complex-formation inhibiting peptide is added to this system, fibrinogen can fall in the state of fibrin monomer analog. Examples of the complex-formation inhibiting peptide used in the present invention include glycyl-prolyl-arginyl-proline, and glycyl-prolyl-arginyl-arginylsacrocine (ANDREW P. et al., Proc. Natl. Sci. USA, 1978).
In the screening in the method for producing the hybridoma of the present invention, antibody-forming cells are selected which are not reactive with either fibrinogen or fibrin decomposition products, and are reactive with the fibrin monomer analogs comprising fibrinogen treated with the snake venom, fibrinogen treated with thrombin, and soluble fibrin analogs obtained by further adding fibrinogen to the aforementioned fibrin monomer analog to become complexed therewith. In this screening step, substances to be screened are substances which are respectively immobilized on a plate for immunoenzyme assays. The fibrinogen treated with thrombin is a substance obtained by treating fibrinogen, immobilized on a plate, with thrombin.
In the cell fusion step in the method for producing the hybridoma of the present invention, if the antibody-forming cell and the myeloma cell are cells which can fuse, the animal of origin is not limited. From the viewpoint of cell fusion efficiency, however, it is preferred to use anti-forming cells and myeloma cells from the same sort of animal. As a cell fusion process, the process according to Kobler and Milstein (Nature 256, 495-497, 1975) can be used to produce a hybridoma producing an antibody having a desired specific reactivity.
The hybridoma of the present invention was deposited as Deposit No. FERM P-16276 with the Ministry of International Trade and Industry, Agency of Industrial Science and Technology, National Institute of Bioscience and Human Technology on Jun. 17, 1997 and transferred as Deposit No.. FERM BP-6386 to International Deposit based on the Budapest Treaty on June 17, 1998. All restrictions upon public access to the deposit will be irrevocably removed upon the grant of a patent on this Application and the deposit will be replaced if viable samples cannot be dispensed by the depository.
The present hybridoma can be cultured in vitro or in vivo to secrete a monoclonal antibody.
The monoclonal antibody of the present invention can be obtained by culturing the hybridoma obtained in the aforementioned manner in a test tube or in an abdominal cavity in an animal. The resultant antibody can be purified by a purifying method such as the protein A method, or ion-exchange chromatography. The immunoassay using the monoclonal antibody of the present invention comprises reacting a specimen containing a fibrin monomer which is present in a body fluid and is not solubilized with an immobilized monoclonal antibody which is not reactive with fibrinogen and is specifically reactive with the fibrin monomer, to assay fibrin in the body fluid. The immunoassay of the present invention is not limited to any special assay, and may be an assay based on any immunological binding using the monoclonal antibody of the present invention. Examples of a specimen to be assayed include any specimen which may contain a fibrin monomer (solubilized fibrin), in particular blood, plasma, serum, and urine.
As the immunoassay method, the following can be used: the EIA method of adsorbing at least one monoclonal antibody obtained according to the present invention onto a polystyrene plate, balls, magnetic particles or the like, adding a specimen thereto, and subsequently using an antibody labeled by an enzyme such as alkaliphosphatase, peroxidase, or alactosidase; the RIA method of using a radioactive isotope labeled antibody; and the FIA (florescent immunoassay) method of using a florescent antibody. Besides, the present invention can be applied to carrier agglutination assays which immobilize at least one antibody obtained according to the present invention on the surface of insoluble carrier particles (liposome, latex or the like) by chemical or physical bonding, mixing the antibody-immobilized carrier with a specimen, and measuring the amount of the resultant coagulation on a slide in or a cell. In this carrier agglutination assay preferably the speed of immunological reaction is measured at least two times as an increase in the absorbance after the reaction starts, so as to assay the amount of a native fibrin monomer or soluble fibrin, which are in a body fluid.